Diagnositc methods for determining susceptibility to convulsive conditions

ABSTRACT

The present invention exploits the discovery that amounts of uracil and thymine metabolites, especially β-aminoisobutyric acid, in various bodily fluids, especially urine, are correlated with the occurrence of epilepsy when compared to matched control subjects. Analytical and diagnostic protocols, including a novel high performance liquid chromatography system, for use in the invention are disclosed.

RELATED APPLICATIONS

[0001] This application claims the priority of U.S. provisional patentapplication No. 60/318,139, filed Sep. 7, 2001, and U.S. provisionalpatent application No. 60/378,781, filed May 7, 2002. The contents ofeach of these aforementioned applications are hereby incorporated hereinby reference.

BACKGROUND OF THE INVENTION

[0002] A variety of clinical methods exist by which a physician isdirected to a diagnosis of the cause of apparent seizures in a patientas either epilepsy or otherwise. For example, routine blood studiesincluding electrolyte and glucose measurements, complete blood counts,and toxin screens may be carried out to assist a physician indetermining a cause of seizures in a patient. Medical imaging, includingCT and MRI, as well as EEG examinations may also yield valuable clinicalinformation in this regard. There are, however, no routinely usedprospective or predictive clinical tests which a physician may performwhich indicate whether or not a patient is at risk of developingseizures in the future.

[0003] Retrospective studies have revealed that several factors areassociated with an increased risk of seizure, for example, a familialhistory of seizures, meningitis, or a recent head trauma. Anindividual's susceptibility to seizure is determined additionally by theindividual's brain chemistry, and consequently a head trauma of equalmagnitude, e.g., may precipitate seizures in one individual, but notanother. Presently, there is no predictive test to distinguish betweenthese two hypothetical individuals.

[0004] To the contrary, following head trauma or insult to the brain itis common practice to administer prophylactically anti-seizure drugs tomost patients who fall into an “at risk” category, without any analysisof the individual's actual risk. Accordingly, patients who are at lowerrisk of developing seizures are subjected to the unnecessaryside-effects of various drugs, such as, e.g., inhibition ofneuroplasticity. A need remains, therefore, for a predictive test whichmore accurately indicates a patient's actual risk of developingseizures.

[0005] Distinguishing pseudoseizures from seizures is another clinicalneed that such a test may address. Pseudoseizures are seizure-likespells with no physiological basis. They can either be intentionally orsubconsciously induced. The treatment for pseudoseizures is oftenpsychological in nature, and patients undergo unnecessary effects ifanticonvulsant medication is administered due to a misdiagnosis.

[0006] Although epileptic seizures are rarely fatal, large numbers ofpatients require medication to avoid the disruptive, and potentiallydangerous, consequences of seizures. In many cases, medication isrequired for extended periods of time, and in some cases, a patient mustcontinue to take prescription drugs for life. Furthermore, drugs usedfor the management of epilepsy have side-effects associated withprolonged usage, and the cost of the drugs can be considerable.

[0007] It has been postulated that free amino acids play a role in thenormal functioning of the central nervous system. Amino acidconcentrations in the brain specifically depend on several factors,including tissue metabolism, blood flow, transport or exclusion at theblood brain barrier, and renal or hepatic function. As such, amino acidimbalances associated with neurological disorders are of interest andhave served as the basis for a variety of investigations.

[0008] However, the findings of previous studies on amino acidimbalances in epilepsy, including those by Plum (Journal ofNeurochemistry 1974, 23, 595-600), Mutani et al. (Epilepsia 1974, 15,595-597), Crawford and Chadwick (Epilepsy Research 1987, 1, 328-338),Haines et al. (Epilepsia 1985, 26, 642-648), Monaco et al. (ItalianJournal of Neurological Sciences 1994, 15, 137-14), van Gelder et al.(Neurochemical Research 1980, 5, 659-671), and Ferrie et al. (EpilepsyResearch 1999, 34, 221-229), are inconsistent. In addition tomethodological sources of variation, inter-study variability has beenattributed to such factors as heterogeneity within the sample populationbeing examined, circadian variation and short-term dietary amino acidintake.

[0009] Anti-epileptic medication may also contribute to inter-studyvariability as increases in glycine, serine and alanine, have been notedupon valproic acid administration, while increases in free and totalβ-aminobutyric acid, homocarosine (a conjugate of β-aminobutyric acid),β-alanine, glycine and β-aminoisobutyric acid occur upon vigabatrinadministration. Alternatively, administration of carbamazepine,ethosuximide and mephobarbital leads to decreases in leucine, prolineand phenylalanine, respectively.

SUMMARY OF THE INVENTION

[0010] The present invention exploits the discovery, described herein,that amounts of uracil and thymine metabolites, especiallyβ-aminoisobutyric acid, in various bodily fluids, especially urine, arecorrelated with the occurrence of epilepsy when compared to matchedcontrol subjects. Analytical and diagnostic protocols, including a novelhigh performance liquid chromatography system, for use in the inventionare disclosed.

[0011] Reported experiments with β-alanine in animals relate toexploiting its neuro-inhibitory effects, e.g. studying how it mitigatesthe extent or threshold of seizure when co-administered with a drugsubstance known to cause seizures. It has not been previouslyrecognized, however, that imbalances of endogenous β-alanine may beindicative of susceptibility to seizure, especially idiopathic seizuresor epilepsy, familial history, and seizures resulting from head trauma.The present method may be used with noninvasive (e.g. urine collection)or minimally invasive techniques (e.g. blood collection). The method ofthe invention may be used to analyze neuro-active molecules such asamino acids in the urine of subjects.

[0012] In particular, the invention relates to methods of diagnosis ofconvulsive conditions or susceptibility thereto in a subject, wherein abodily fluid from a subject is analyzed for the presence of aneuro-active molecule associated with a convulsive condition, and thesubject is diagnosed as at risk of a convulsive condition orsusceptibility thereto if the amount of the compound indicates alikelihood of same in the subject. Preferred neuro-active moleculesinclude metabolites of uracil and thymine, particularly β-amino acids,preferably β-aminoisobutyric acid.

[0013] Furthermore, the invention relates to methods of modulating,including inhibiting or preventing, the onset of a convulsive conditionin a subject, wherein a bodily fluid from a subject is analyzed for thepresence of a neuro-active molecule associated with a convulsivecondition; determining from the amount of the compound in the bodilyfluid whether the subject is at risk of a convulsive condition; andtreating the subject, if at risk of a convulsive condition, to modulatethe onset of the convulsive condition in the subject. Preferredneuro-active molecules include metabolites of uracil and thymine,particularly β-amino acids, preferably β-aminoisobutyric acid.

[0014] Additionally, a method of quantifying neuro-active molecules suchas β-alanine or β-aminoisobutyric acid is described, comprisingcollecting and optionally deproteinizing a bodily fluid sample, e.g.urine, derivatizing the amino acids present in the (deproteinized)sample, and analyzing the (derivatized) amino acids by chromatography(such as reversed phase high performance liquid chromatography), thechromatography system comprising a column, mobile phases (preferablyacetate buffer and methanol), an optional internal standard (preferablyD,L-ethionine) and a set of external standards of varying concentration,and a separation program which produces a resolution for each of theneuro-active molecules of interest with all other amino acids andmolecules present in the bodily fluid of equal to or greater than one.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 illustrates various metabolic pathways implicated in themedical conditions described herein and related to β-alanine andβ-aminoisobutyric acid.

[0016]FIG. 2 shows a representative chromatogram depicting the elutionprofile for a 100 μmol/L standard mixture of 23 amino acids according toa method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention entails a method of diagnosing a convulsivecondition or susceptibility thereto in a subject comprising the steps ofanalyzing a bodily fluid from a subject for the presence or amount(s) ofone or more neuro-active molecule(s), or the relative amounts ofneuro-active molecules (e.g. ratio), associated with a convulsivecondition; and diagnosing the subject as at risk of a convulsivecondition or susceptibility thereto if the amount of said compoundindicates a likelihood of same in said subject. Said subject need nothave actually developed seizures.

[0018] According to the invention, a standard against which the abovemeasure or measures from test bodily fluids are compared may be dataobtained from a data bank corresponding to currently accepted normallevels of neuro-active molecules under analysis. In situations such asthose where standard data are not available, the methods of theinvention may further comprise conducting corresponding analyses in asecond set of one or more biological samples known not to be at risk ofa convulsive condition or susceptibility thereto. Such additionalbiological samples could be obtained, for example, previously from thesubject under consideration, or from unaffected members of the public.

[0019] According to the methods of the invention, the comparison of theabove measure or measures may be a straight-forward comparison, such asa ratio, or it may involve weighting of one or more of the measures,relative to, for example, their importance to the particular situationunder consideration. The comparison may also involve subjecting themeasurement data to any appropriate statistical analysis. In mostdiagnostic procedures in accordance with the invention, one or morebiological samples obtained from an individual will be subjected to abattery of analyses in which any number of neuro-active molecules aresought to be detected. In any such diagnostic procedure it is possiblethat one or more of the measures obtained will render an inconclusiveresult; accordingly, data obtained from a battery of measures is likelyto provide for a more conclusive diagnosis. It is for this reason thatan interpretation of the data based on an appropriate weighting schemeor statistical analysis is desirable.

[0020] The term “convulsive disorder” or “convulsive condition”according to the invention includes conditions wherein a subject suffersfrom convulsions. Convulsive disorders include, but are not limited to,epilepsy, ictogenesis, epileptogenesis, and non-epileptic convulsions,and convulsions due to administration of a convulsive agent or trauma tothe subject.

[0021] A seizure is a single discrete clinical event caused by anexcessive electrical discharge from a collection of neurons through aprocess termed “ictogenesis.” As such, a seizure is merely the symptomof epilepsy.

[0022] Epilepsy is a dynamic and often progressive process characterizedby an underlying sequence of pathological transformations whereby normalbrain is altered, becoming susceptible to recurrent seizures through aprocess termed “epileptogenesis.” While it is believed that ictogenesisand epileptogenesis have certain biochemical pathways in common, the twoprocesses are not identical.

[0023] Ictogenesis (the initiation and propagation of a seizure in timeand space) is a rapid and definitive electrical/chemical event occurringover seconds or minutes. Epileptogenesis (the gradual process wherebynormal brain is transformed into a state susceptible to spontaneous,episodic, time-limited, recurrent seizures, through the initiation andmaturation of an “epileptogenic focus”) is a slow biochemical orhistological process which generally occurs over months to years.

[0024] Epileptogenesis is a two phase process: Phase 1 epileptogenesisis the initiation of the epileptogenic process prior to the firstseizure, and is often the result of stroke, disease (e.g. meningitis),or trauma, such as an accidental blow to the head or a surgicalprocedure performed on the brain. Phase 2 epileptogenesis refers to theprocess during which a brain that is already susceptible to seizures,becomes still more susceptible to seizures of increasing frequency orseverity. While the processes involved in epileptogenesis have not beendefinitively identified, some researchers believe that up-regulation ofexcitatory coupling between neurons, mediated by N-methyl-D-aspartate(NMDA) receptors, is involved. Other researchers implicatedown-regulation of inhibitory coupling between neurons, mediated byγ-aminobutyric acid (GABA) receptors, pre- or post-synaptically.

[0025] The term “subject” includes animals susceptible to convulsivedisorders, epileptogenesis or capable of suffering fromepileptogenic-associated states, such as warm-blooded animals, morepreferably a mammal, including, e.g. non-human animals such as rats,mice, cats, dogs, sheep, horses, cattle, in addition to humans. In apreferred embodiment, the subject is a human. Subjects with a familyhistory of convulsive conditions, a history of cerebral hypoxia orischemia, intracranial hemorrhage, central nervous system infection ordisease, drug or alcohol withdrawal, fever, trauma, brain tumor,cerebrovascular disease, metabolic disorder, degenerative centralnervous system disease, drug or alcohol addiction or use, uremia,hepatic dysfunction, hypoglycemia, epilepsy, or seizure are preferredsubjects for analysis according to the invention because they may be atrisk for convulsions. Additionally, preferred subjects include those whohave recently been administered an antibiotic, anesthetic, analgesic,immunomodulatory, psychotropic, sedative, radiographiccontrast-enhancing, stimulant or hallucinogenic drug. A particularlypreferred subject according to the invention is one who has suffered ahead trauma and is at risk of developing post-traumatic epilepsy (PTE).

[0026] A seizure or convulsion, which terms may be used interchangeablyherein, may be complex partial, simple partial, absence, secondarygeneralized tonic clonic, primary generalized tonic clonic, myoclonic,or atonic.

[0027] “Bodily fluid” as used herein includes, e.g., urine, blood, bloodserum, amniotic fluid; cerebrospinal (i.e. CSF) and spinal fluid,synovial fluid, conjunctival fluid, salivary fluid, vaginal fluid,stool, seminal fluid, lymph, bile, tears, and sweat. A bodily fluid isadvantageously CSF, urine, or blood or its components parts, e.g plasma.A particularly preferred bodily fluid is urine.

[0028] “Neuro-active molecules” according to the invention includeneurotransmitters, such as amino acid neurotransmitters,neutrostimulators, and neurodepressants. Such neuro-active molecules mayalter the ability of a nerve cell to depolarize or to release or take upneurotransmitter molecules. As described herein, preferred neuro-activemolecules of the invention include metabolites of uracil or thymine,especially β-amino carboxylic acids (comprising at least thesub-structure N—C—C—(C═O)—O) such as β-alanine and β-aminoisobutyricacid, and derivatives thereof. Such derivatives may be esters or otherbioconjugates (including glucuronic acid and sterol conjugates).

[0029] The invention relates to convulsive conditions related to thymineor uracil metabolism, including abnormalities thereof, and therefore thecompounds depicted in FIG. 1 are neuro-active molecules according to theinvention as described further herein below.

[0030] “Analyzing” as used herein may be any step which eitherqualitatively or quantitatively indicates the amount or presence of aneuro-active molecule. Examples of analyses of the present inventioninclude chromatography (including high-performance liquidchromatography, thin layer chromatography, or gas chromatography),spectroscopy, spectrometry, and colorimetry (such as by use of acolor-changing indicator as in, for example, a “dip stick” or “teststrip” as commonly used in the detection of glucose in urine), althoughother functional equivalents may be employed.

[0031] An analysis step may include further steps of preparing a samplefor study, such as removal of interfering compounds (i.e.non-neuro-active molecules) from the bodily fluid by such means asprecipitation, filtration, and the like. Additionally, neuro-activemolecules may be derivatized prior to analysis to facilitate detection.For example, in analysis protocols where detection is by absorption, itmay be advantageous to covalently attach a chromophore to theneuro-active molecules.

[0032] The present invention also relates to a method of modulating theonset of a convulsive condition in a subject comprising the steps ofanalyzing a bodily fluid from a subject at risk of a convulsivecondition for the presence of a neuro-active molecule associated with aconvulsive condition; determining from the amount of said compound insaid bodily fluid whether said subject is at risk of a convulsivecondition; and treating said subject, if at risk of a convulsivecondition, to modulate the onset of said convulsive condition in saidsubject.

[0033] “Modulating” means altering the likelihood that a seizure willoccur. Generally, modulating will mean reducing or inhibiting thelikelihood of a future seizure in a subject in accordance with theinvention. Modulating may refer to any convulsive condition or aprecursor thereof.

[0034] The terms “treatment,” “treating,” or “treat,” include theadministration of an agent (e.g. an anticonvulsive oranti-epileptogenic, prophylactic or therapeutic pharmaceuticalcomposition) to a subject, who has a disease or disorder, a symptom of adisease or disorder, or is at risk of suffering from the disease ordisorder in the future, such that the disease or disorder (or at leastone symptom of the disease or disorder) is cured, healed, prevented,alleviated, relieved, altered, remedied, ameliorated, improved orotherwise affected, preferably in an advantageous manner. “Agents”include anti-convulsive, anti-seizure, or anti-epileptogenic agents,such as described in U.S. Pat. No. 6,306,909 B1. Such a treatment stepmay comprise administering an effective amount of an anti-convulsive,anti-seizure, or anti-epileptogenic pharmaceutical composition.

[0035] The language “effective amount” of a compound is that amountnecessary or sufficient to treat or prevent a particular condition,e.g., to prevent the various morphological and somatic symptoms of anepileptogenic-associated state. The effective amount can vary dependingon such factors as the size and weight of the subject, the type ofcondition, or the particular agent. For example, the choice of thepharmaceutical composition can affect what constitutes an “effectiveamount.” One of ordinary skill in the art would be able to study theaforementioned factors and make the determination regarding theeffective amount of the pharmaceutical composition without undueexperimentation.

[0036] The term “anti-epileptogenic agent” includes agents which arecapable of inhibiting epileptogenesis, e.g., suppressing the uptake ofsynaptic GABA (e.g., blocking GABA transporters, e.g. GAT-1, GAT-2 orGAT-3), depressing glutamatergic excitation (e.g., interacting with anNMDA receptor, e.g. at the strychnine-insensitive glycine co-agonistsite), binding to a GABA receptor (e.g. GABA_(A)), altering (e.g.,increasing or suppressing) the metabolism of GABA (e.g, via inhibitionof GABA transaminase).

[0037] Further examples of pharmaceutical compositions of the presentinvention include carbamazepine, clobazam, diazepam, lamotrigine,lorazepam, oxazepam, phenobarbital, phenytoin, primidone, valproate,ethosuximide, topirimate, felbamate, clonazepam, clobazam, nitrazepam,vigabatrin, gabapentin, levetiracetam, or tiagabine, or otherpharamceuticals approved for the treatment of seizures or epilepsy bygovernment regulatory agencies (such as the United States Food & DrugAdministration), or combinations thereof.

[0038] Generally, a convulsive condition is selected from the groupconsisting of epileptogenic associated disorders, epileptogenesis, andnon-epileptic convulsions. Inhibiting epileptogenesis includes bothpartial and complete reversal of epileptogenesis. Inhibitingepileptogenesis includes prevention of epileptogenesis or a decrease orslowing in the rate of epileptogenesis (e.g. a partial or complete stopin the rate of epileptogenic transformation of the brain or centralnervous system tissue). It also includes any inhibition or slowing ofthe rate of the biochemical processes or events which take place duringPhase 1 or Phase 2 epileptogenesis and lead to epileptogenic changes intissue, i.e., in tissues of the central nervous system (CNS), e.g. thebrain. Examples of processes in pathways associated with epileptogenesisare discussed in more detail herein. Modulating epileptogenesis alsoincludes the prevention, slowing, halting, or reversing the process ofepileptogenesis, i.e., the changes in brain chemistry which result inepileptic seizures.

[0039] The term “epileptogenic-associated disorders” includes disordersof the central and peripheral nervous system which may advantageously betreated as described in, e.g. U.S. Pat. No. 6,306,909 B1 and PCTpublication WO 98/40,055. In an advantageous embodiment, the nervoussystem disorders are disorders associated with or related to the processor the results of epileptogenic transformation of the brain or othernervous tissue.

[0040] Examples of epileptogenic-associated disorders include epilepsy,head trauma, stroke, multiple sclerosis, amyotrophic lateral sclerosis,psychoses, cerebral ischemia, motor neuron disease, Alzheimer's disease,encephalitis (including encephalitis arising from chicken-pox, measlesor pertussis), infections of the CNS (meningitis, encephalitis),subdural haematoma, brain tumour, and birth defects including anoxicbrain injury, dementia and other disorders (in humans or animals) inwhich altered activity of neurotransmitters is a cause, at least inpart, of the disorder (see, e.g. Schoepp et al., Eur. J. Pharmacol.1991, 203, 237-243; Leeson et al., J. Med. Chem. 1991, 34, 1243-1252;Kulagowski et al., J. Med. Chem. 1994,37, 1402-1405; Mallamo et al., J.Med. Chem. 1994, 37, 4438-4448; and references cited therein). The termepileptogenic-associated disorders includes both convulsive disordersand disorders associated with NMDA receptor activity.

[0041] The invention also relates to particular novel methods ofanalysis, including a method of quantifying neuro-active molecules, suchas β-alanine or β-aminoisobutyric acid, comprising the steps ofcollecting a bodily fluid sample, such as urine; optionallydeproteinizing said sample; optionally derivatizing the neuro-activemolecules present in said (deproteinized) sample; and analyzing said(derivatized) neuro-active molecules by chromatography, saidchromatography system comprising a column (preferably a reversed phaseC8 or C18 column), mobile phases (preferably acetate buffer andmethanol), an optional internal standard (preferably D,L-ethionine) anda set of external standards of varying concentration, and a separationprogram which produces a resolution for derivatized neuro-activemolecule(s) of interest present in said sample of equal to or greaterthan one. This method is most advantageously applied to the analysis ofamino acid neuro-active molecules, particularly β-aminoisobutyric acidor another metabolite of uracil or thymine.

[0042] The analysis method may further comprise a step of deproteinizingsaid bodily fluid, for example by ultrafiltration, ultracentrifugation,or chemical precipitation. The chemical precipitation step may employ aprecipitating agent, for example, sulfosalicylic acid, perchloric acid,trichloroacetic acid, picric acid, acetonitrile, ethanol, acetone, ormethanol.

[0043] The derivatizing step may covalently attach a chromophore to anamino acid, and preferred reagents for use in such a derivatizing stepinclude o-phthalaldehyde, 9-fluorenylmethylchloroformate, phenylisothiocyanate, or 1-dimethylaminonaphthalene-5-sulphonyl chloride, aswell as other commercially available reagents.

[0044] In this invention, levels of β-aminoisobutyric acid arecorrelated with the occurrence of epilepsy, as demonstrated by thefollowing, wherein the concentrations of γ-alanine and its metabolicequivalent β-aminoisobutyric acid in urine collected from subjects withepilepsy and matched control subjects were studied. A novelreversed-phase high performance liquid chromatography (RP-HPLC) programis disclosed for the analytical separation and quantification ofβ-alanine and β-aminoisobutyric acid in urine.

[0045] Protein in physiological fluids is typically inevitable, but itinterferes with amino acid analysis and shortens the lifetime ofchromatographic columns. Several methods have been proposed for theremoval of protein from physiological fluids, including chemicalprecipitation, ultrafiltration, and ultracentrifugation, with chemicalprecipitation finding the most frequent use. Sulfosalicylic acid is acommon precipitation agent, and is used as a solution in distilledde-ionized water in concentrations as high as 20% (w/v). Thismethodology does, however, tend to lower the concentrations of asparticacid and glutamic acid in solution due to their decreased solubilityunder strongly acidic conditions. Once precipitation is complete, thesample is filtered, making it ready for derivatization and analysis.

[0046] With a few exceptions, free amino acids cannot be detected usingexperimental techniques such as UV absorption. A variety of methodsknown in the art are therefore available for the derivatization of aminoacids prior to analytical separation and detection by RP-HPLC.Generally, pre-column derivatization is preferred, as it results inincreased resolution and sensitivity over the corresponding post-columnmethodology. With derivatization using o-phthalaldehyde (OPA),9-fluorenylmethyl chloroformate (FMOC-Cl), phenyl isothiocyanate (PITC)or 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl-Cl), theautomated OPA method is generally the most amenable to routine analysisof primary amino acids except cysteine.

[0047] With the OPA method, primary amino acids are reacted witho-phthalaldehyde in the presence of the reducing agentβ-mercaptoethanol. Detection of the corresponding derivatized amino acidis achieved by the monitoring of absorbance at a wavelength of 340 nm.This reaction occurs in a 1:1:1 ratio, with the elimination of twomolecules of water, to yield the corresponding fluorescent1-alkylthio-2-alkyl-substituted isoindoles. OPA is inherentlynon-fluorescent and as such gives low reagent interference. Thederivatization reaction is rapid and occurs readily at ambienttemperature. Typically, detection limits lie in the low picomole range.Disadvantages, however, include the instability of the fluorescentisoindoles, the inability to detect secondary amino acids such asproline and hydroxyproline, and the poor fluorescent response arisingfrom the derivatization of a few amino acids, in particular cysteine.

[0048] Several modifications may improve the sensitivity andreproducibility of this reaction as well as the stability of itsproducts. The addition of BRIJ-35 (polyoxyethylene lauryl ether (ICIAmericas)) enhances the fluorescent response of lysine andhydroxylysine. Alternative thiol containing reagents, such as tert-butylthiol, also increase the stability of the corresponding isoindoles. Thereproducibility of the derivatization reaction can be enhanced throughthe maintenance of constant reaction times and temperatures, e.g. viaautomated derivatization at temperature-controlled conditions. Finally,avoiding the use of excess o-phthalaldehyde reagent minimizes thedegradation of the isoindole intermediate, which improves thesensitivity of quantitative amino acid analysis.

[0049] Traditionally, analytical separation and detection of amino acidsin physiological fluids involved ion-exchange chromatography incombination with post-column ninhydrin derivatization and subsequentdetection using either spectrophotometry or colorimetry. However, theadvantage of reduced analysis times, improved resolution and enhancedsensitivity, along with the development of HPLC has prompted a shiftaway from this classical method of amino acid analysis. Several manualand automated RP-HPLC procedures using o-phthalaldehyde derivatizationhave since been developed for the separation and detection of aminoacids in physiological fluids, but very few have been specificallydesigned for the detection and quantification of β-alanine andβ-aminoisobutyric acid.

[0050] Metabolic pathways involving β-alanine and β-aminoisobutyric acidare depicted in FIG. 1. As illustrated in this FIG. 1, β-alanine andβ-aminoisobutyric acid are believed to be endogenously derived via themetabolism of uracil and thymine, respectively.

[0051] The first of three enzymes involved in this pathway isdihydropyrimidine dehydrogenase. This enzyme is responsible forcatalyzing the reversible NAPDH-dependent conversion of uracil andthymine to dihydrouracil and dihydrothymine. This initial step is ratedetermining with respect to the overall breakdown of uracil and thymineto β-alanine and β-aminoisobutyric acid.

[0052] Further transformation of these metabolites, through the actionof dihydropyrimidinase, reversibly yields β-ureidopropionate andβ-ureido-iso-butyric acid.

[0053] Finally, β-alanine synthase, also referred to asβ-ureidopropionase or N-carbamoyl-β-alanine amidohydrolase, facilitatesthe irreversible hydrolytic cleavage of β-ureidopropionate andβ-ureido-iso-butyric acid to give β-alanine and β-aminoisobutyric acidas well as the release of ammonia (NH₃) and carbon dioxide (CO₂). Thisenzyme is of particular importance in animals in that it is directlyresponsible for the in vivo biosynthesis of β-alanine, with its actionoccurring predominantly in the liver. It is the R-isomer ofβ-aminoisobutyric acid that is formed via this reaction. Thecorresponding S-isomer is generated through the metabolism of L-valine.

[0054] Minor sources of β-alanine arise from the actions of two enzymes,aspartate decarboxylase, found in bacteria of the intestinal lumen whichdecarboxylates aspartic acid to give β-alanine; and carnosinase, whichcatabolizes carnosine to give β-alanine and histidine.

[0055] Dihydropyrimidine dehydrogenase is the rate-limiting enzyme inthe catabolic pathway from uracil and thymine to β-alanine andβ-aminoisobutyric acid. A deficiency of this enzyme has deleteriousphysiological effects. Dihydropyrimidine dehydrogenase deficiencyresults from the autosomal recessive inheritance of a mutant allelecoding for the dihydropyrimidine dehydrogenase enzyme (Gonzales, et al.,T.I.P.S. 1995, 16, 325-327). The presence of this mutation leads to theloss of a 165 base pair exon, resulting in the expression of truncatedmRNA. This mutation can lead to a drop in enzyme activity by as much as98 to 100%.

[0056] Dihydropyrimidine dehydrogenase deficiency has two distinctclinical forms (Scriver, et al., The Metabolic and Molecular Bases ofInherited Disease; 7 ed.; Scriver, et al., Eds.; McGraw-Hill, Inc.: NewYork, 1995; Vol. 1). The genetic form, involving an inborn error ofmetabolism, is an early onset disorder commonly associated withneurological signs such as seizures, impaired cognitive development,hypertonia, hyperreflexia, microcephaly and dysmyelination. Theiatrogenic form, which occurs following exposure to the cancerchemotherapeutic agent 5-fluorouracil, is characterized by clinicalsymptoms such as encephalopathy, neurotoxicity and neutropenia.Withdrawal of this drug eliminates all symptoms of this disorder(Tuchman, et al., New Eng. J Med. 1985, 313, 245-249).

[0057] The pathophysiology underlying the association of epilepsy withdihydropyrimidine dehydrogenase deficiency remains unclear. It has,however, been suggested that seizure etiology may arise at the level ofthe nucleic acids (Braakhekke, et al., J. Neuro. Sci. 1987, 78, 71-77).Uridine, a pyrimidine nucleoside, has been shown to exhibitanticonvulsant activity in animal models of epilepsy. Atypicalregulation of uridine and its related compounds may therefore beimportant in explaining abnormal central nervous system regulation. Acorrelation between the lack of β-alanine and the neurological symptomsof dihydropyrimidine dehydrogenase deficiency has also been suggested.

[0058] Since its initial detection, several cases of dihydropyrimidinedehydrogenase deficiency have been documented (van Gennip, et al., Adv.Exp. Med. Biol. 1989, 253A, 111-118). Diagnosis of this disorder isnormally based on presence of high levels of uracil, thymine and5-hydroxymethyluracil (a metabolite of thymine), in physiological fluids(Valik, et al., Mayo Clin. Proc. 1997, 72, 719-725; van Gennip, et al.,Clin. Chem. 1993, 39, 380-385). In urine, uracil and thymine levels canbe elevated by as much as one hundred fold for the iatrogenic form andone thousand fold for the genetic form. Definitive diagnosis of thisdisorder does, however, require conclusive proof of an enzymedeficiency. To quantify dihydropyrimidine dehydrogenase activity,cultured fibroblasts are first incubated with 14C-thymine. The loss of14C-thymine and the formation of 14C-dihydrothymine are then quantifiedusing a combination of HPLC and liquid scintillation counting to give ameasure of enzyme activity (Bakkeren, et al., Clin. Chim. Acta 1984,140, 247-256).

[0059] Dihydropyrimidinuria is a disorder resulting from a deficiency indihydropyrimidinase, the second of three enzymes along the catabolicpathway from uracil and thymine to β-alanine and β-aminoisobutyric acid.Excretion of large quantities of dihydrouracil and dihydrothymine aretherefore associated with this condition. Although it is believed to beautosomal recessive, little else is known about this disorder. Only twocases of dihydropyrimidinuria have been reported. One subject exhibitedconvulsions, lowered consciousness and metabolic acidosis, while theother showed signs of gross microcephaly, spastic quadriplegia,choreiform movements and severe developmental retardation (Webster, etal., The Metabolic and Molecular Bases of Inherited Disease; 7 ed.;Scriver, et al., Eds.; McGraw-Hill, Inc.: New York, 1995; Vol. 2).

[0060] Catabolism of β-alanine primarily occurs through the actions oftwo aminotransferases, β-alanine-α-ketoglutarate transaminase andβ-alanine-pyruvate transaminase, and results in the production ofmalonic acid semialdehyde. It is a deficiency of the former enzyme thatis believed to underlie hyper-β-alaninemia, a rare disordercharacterized by increased levels of β-alanine and GABA in cerebrospinalfluid, plasma and urine as well as β-aminoisobutyric acid in urine(Scriver, et al., New Eng. J. Med. 1966, 274, 635-643). This postulateis supported by three key observations. First, the administration ofpyridoxine, whose derivative pyridoxal-5-phosphate acts as anaminotransferase coenzyme, has been effective in the symptomatictreatment of hyper-β-alaninemia. Second, β-alanine, S-β-aminoisobutyricacid, and GABA are all transaminated with α-ketoglutarate via theactions of these transaminases in both the brain and liver. Finally, thefact that both β-alanine and GABA are elevated in physiological fluidssuggests a lack of involvement of β-alanine-pyruvate transaminase, whosesubstrate specificity is for β-alanine alone.

[0061] Only two cases of hyper-β-alaninemia have been reported to date.One subject exhibited somnolence and repeated grand-mal seizures anddied within five months of birth, while the other was described ashaving intermittent generalized tonic-clonic seizures, lethargy andCohen's syndrome (Higgins, et al., Neurology 1994, 44, 1728-1732). Theetiology of these neurological symptoms remains unclear. Plausibleexplanations include the inhibition of GABA transaminase by excessβ-alanine, competitive binding of β-alanine to the GABA receptor, aswell as agonism of the strychinine-sensitive glycine and NMDA receptorsby β-alanine.

[0062] Hyper-β-aminoisobutyric aciduria is a reasonably prevalentdisorder involving a deficiency in β-aminoisobutyrate-pyruvatetransaminase, an enzyme responsible for the catabolism ofβ-aminoisobutyric acid. Subjects with this disorder typically exhibitless than 10% of the normal enzyme activity and therefore excrete largequantities of this amino acid. The genetic variant of this disorder ispostulated to be recessive, stemming from a genetic polymorphism at asingle locus. This trait appears to have a nonrandom distribution withinthe population, with highest frequencies in the Micronesian populationand lowest frequencies in the Caucasian population.

[0063] Other factors are known to influence the excretion ofβ-aminoisobutyric acid. Children are known to have higher excretionrates than adults, while females tend to have higher excretion ratesthan males. Enhanced excretion of β-aminoisobutyric acid is also afactor to be considered with neoplastic states and with Down's syndromeas well as during periods of increased somatic cell growth, whenpyrimidine turnover is significantly elevated.

[0064] Therefore, a variety of neuro-active molecules, such as aminoacids, including β-alanine, β-aminoisobutyric acid, and those compoundsdepicted in FIG. 1 are within the scope of the present invention.

[0065] The invention described herein is exemplified by the followingnon-limiting method. Other analytical methods known in the art may beemployed according to the teachings herein. The method described belowmay be modified by one skilled in the art using no more than routineexperimentation. Such functionally equivalent analytical methods arealso encompassed by the instant invention.

[0066] HPLC-grade methanol and HPLC-grade glacial acetic acid wereobtained from Fisher Scientific (Fair Lawn, N.J.), sodium acetate wasobtained from Sigma-Aldrich (Milwaukee, Wis.) and fluoraldehyde reagentsolution was obtained from Pierce Chemical (Rockford, Ill.). IndividualL-amino acids, β-alanine, β-aminoisobutyric acid, D,L-ethionine andsulfosalicylic acid were also obtained from Sigma-Aldrich.

[0067] A System Gold liquid chromatographic system was combined with aModel 125 programmable solvent module fitted with an Altex 210Ainjection valve, a Model 166 programmable UV-VIS detector module(Beckman, San Ramon, Calif.); a Dell 489P/33 computer and an EpsonFX-870 printer. A 5 μm Ultrasphere ODS column (250 mm×4.6 mm I.D.) witha 5 μm Ultrasphere ODS guard column (45 mm×4.6 mm I.D.), both fromBeckman, were used. Solvents were filtered through 0.22 μm nylon filters(N02SP04700) from Osmonics (Minnetonka, Minn., U.S.A.). Distilledde-ionized water was prepared using a Culligan de-ionizer fromStructural Fibers (Chardon, Ohio).

[0068] Human subjects with epilepsy and matched control subjects weresub-classified into the following five groups:

[0069] Epilepsy Groups: E+D+) subjects whose seizure frequency per monthwas greater than zero (mean average seizure frequency per month 1.23;range 0.2-4) over the past six months prior to sample collection and whowere taking anti-epileptic medication (8 male and 7 female), E−D+)subjects whose seizure frequency per month was zero over the past sixmonths prior to sample collection and who were taking anti-epilepticmedication (13 male and 9 female), and E−D−) subjects whose seizurefrequency per month was zero over the past six months prior to samplecollection and who were not taking anti-epileptic medication (9 male and3 female).

[0070] Control Groups: C−D+) subjects without a prior history ofseizures and who were taking anti-epileptic medication (18 male and 6female) and C−D−) subjects without a prior history of seizures and whowere not taking anti-epileptic medication (22 male and 5 female).

[0071] For those subjects with epilepsy, the etiology of seizures wasinfection (chicken-pox, measles encephalitis, pertussis encephalitis,viral encephalitis, viral meningitis) for 8 subjects, trauma (subduralhematoma) for 2 subjects, birth complications (anoxic brain injury) for11 subjects, central nervous system defects (cerebral cortical atrophywith hydrocephalus, congenital brain abnormality with hydrocephalus,spastic quadriplegia, spina bifida with hydrocephalus) for 7 subjects,prenatal complications (maternal congenital rubella, maternal eclampsia)for 2 subjects, miscellaneous (fetal complications due to hyperemesisgraviderum, Rett syndrome) for 2 subjects and unknown for 17 subjects.

[0072] Subjects with epilepsy in groups E+D+ and E−D+ as well as matchedcontrol subjects in group C−D+ were receiving various combinations ofanti-epileptic medication including carbamazepine, clobazam, diazepam,lamotrigine, lorazepam, oxazepam, phenobarbital and phenytoin. Elevensubjects with epilepsy (30%) and 21 control subjects (88%) were takingonly one medication, 14 subjects with epilepsy (38%) and three matchedcontrol subjects (12%) were taking two medications, 10 subjects withepilepsy (27%) were taking three medications, while 2 subjects withepilepsy (5%) were taking four medications.

[0073] Subjects with one of primary generalized epilepsy (absenceseizures), chromosomal abnormalities (fragile X syndrome, Down'ssyndrome, and Angelman's happy puppet syndrome) or amino acid disorders(phenylketonuria, amino aciduria) were excluded, as were subjectsreceiving the anti-epileptic medication vigabatrin.

[0074] A single urine sample was collected from each subject in thestudy population between the hours of 6:00 and 11:00 am on the morningfollowing a meatless dinner. In this way, it was anticipated that samplevariability arising from circadian variation as well as the influence ofdiet on uracil concentrations might be minimized. Samples were screenedfor leukocytes, nitrite, pH, protein, glucose, ketones, urobilinogen,bilirubin and blood using a CHEM 9 Chemstrip Urine Test Strip fromBoehringer Mannheim (Indianapolis, Ind., U.S.A). All samples used inthis study fell within normal reference ranges for the aforementionedcriteria. Each sample was separated into three aliquots and placed inseparate labeled disposable vials. Samples were cooled to 0-4° C. fortransport. For long-term storage, samples were kept at −50 to −60° C.

[0075] A stock solution of β-alanine and β-aminoisobutyric acid wasprepared by dissolving each compound in distilled de-ionized water to afinal concentration of 0.1 mmol/L. Working external standards wereprepared from the stock solution by dilution with distilled de-ionizedwater to final concentrations of 1, 5, 10, 20, 40, 60, 80, 100, 200,400, and 600 μmol/L. A stock solution of D,L-ethionine, the internalstandard, was prepared by dissolving it in distilled de-ionized water toa final concentration of 1 mmol/L. A stock solution of 23 amino acidswas prepared by dissolving each compound in distilled de-ionized waterto a final concentration of 1 mmol/L. Stock solutions of the individualL-amino acids were prepared to final concentrations ranging from 30μmol/L to 2.5 mmol/L. The standard amino acid mixture was used formethod development while individual L-amino acid standards were used forpeak identification. For long-term storage, all standards were kept at−50° C. to −60° C.

[0076] Urine samples were deproteinized via chemical precipitation usingsulfosalicylic acid according to the following procedure, although otherdeproteinization procedures as known in the art may also be employed.

[0077] Urine samples were thawed and vortex mixed for 30 sec. In adisposable tube, 100 μL 15% (w/v) sulfosalicylic acid was added to 1 mLof sample. The resulting mixture was vortex mixed and left standing for5 min. The treated sample was filtered through a disposable 5″ P.P.chromatography column fitted with a medium (45-90 μm) filter from DiaMedLab Supplies (Mississauga, ON, Canada) into a second disposable tube.Deproteinization led to urine sample dilution by a factor of 1.10.

[0078] Fluoraldehyde reagent solution, containing o-phthalaldehyde (0.8mg/mL, purchased from Pierce Chemical Co., Rockford, Ill.),β-mercaptoethanol and BRIJ-35, in a borate buffer (pH˜10), was used forthe derivatization of primary amino acids in urine samples and standardsaccording to the following procedure: In an ice bath, 10 μL of 1 mmol/LD,L-ethionine (internal standard) was added to 30 μL of a sample,external standard or blank (distilled de-ionized water) in a disposabletube. The resulting solution was vortex mixed for 10 sec. To this wasadded 20 μL of fluoraldehyde reagent solution and the resulting solutionwas vortex mixed for 20 sec. After 1 min, 80 μL of 0.1 mmol/L sodiumacetate buffer (pH=7.0) was added and the solution was vortex mixed for20 sec. The derivatized solution was loaded onto the injection loop and20 μL was injected at 2 min.

[0079] RP-HPLC was used to separate and detect the presence of aminoacids in urine. Eluent A (50 mmol/L sodium acetate buffer, pH 5.7) andeluent B (methanol) were degassed by vacuum filtration through a nylonfilter (0.22 um). The upper and lower pressure limits were set at 4.00and 0.00 kPSI. For all samples and standards, the injection volume was20 μL. Elution was performed at ambient temperature at a flow rate of1.5 mL/min with the concentration of eluent B as follows: 0-16 min,isocratic elution at 30%: 16-21 min, linear gradient from 30-36%: 21-28min, isocratic elution at 36%: 28-32 min, linear gradient from 36-55%:32-35 min, isocratic elution at 55%: 35-38 min, linear gradient from55-70%: 38-48 min isocratic elution at 70%. The absorbance of the columneluate was monitored at a wavelength of 340 nm. The integrationparameters, peak threshold and peak width, were set at 3.9×10⁻⁴ and0.48, respectively. The column was washed and reconditioned with theconcentration of eluent B as follows: 48-51 min, linear gradient from70-100%: 51-56 min, isocratic elution at 100%: 56-59 min, lineargradient from 100-30%: 59-69 min, isocratic elution at 30%. Totalanalysis time per sample was 48 min, while total analysis time betweensamples was 69 min.

[0080] The RP-HPLC procedure was optimized to ensure maximal andreproducible separation of a 23 amino acid mixture with respectableresolution of taurine, β-alanine, β-aminoisobutyric acid andβ-aminobutyric acid. A representative chromatogram depicting the elutionprofile for a 100 μmol/L standard mixture of 23 amino acids isillustrated in FIG. 2.

[0081] Urinary creatinine concentrations were quantified and used tostandardize amino acid results for the sample population with respect torenal clearance. β-Alanine and βaminoisobutyric acid concentrations wereexpressed as ratios relative to the concentrations of cretinine in theirrespective samples. TABLE 1 Levels of β-alanine and β-aminoisobutyricacid in urine from subjects with epilepsy. β-Alanine^(a)β-Aminoisobutyric Acid^(a) Subject (μmol/mmol Creatinine) (μmol/mmolCreatinine)  1 1.400 41.85  2 0.5799 39.72  3 1.411 11.80  4 2.061 10.63 5 1.220 16.95  6 0.8061 5.660  7 7.092 101.2  8 0.1816 10.35  9 1.751<0.3310 10 <0.2967 34.80 11 4.061 125.3 12 2.217 28.31 13 1.371 30.29 144.160 6.415 15 <0.1312 7.183 16 1.852 42.52 17 5.307 75.77 18 2.46516.19 19 1.080 9.256 20 1.143 7.380 21 <0.3580 9.930 22 3.587 140.1 230.9659 345.7 24 0.9704 7.048 25 1.017 18.57 26 14.39 43.09 27 2.4649.337 28 0.4409 13.98 29 1.495 42.77 30 1.233 71.01 31 0.7048 16.58 322.008 42.58 33 11.06 12.75 34 3.926 15.36 35 5.954 23.07 36 0.6052 3.42837 0.2621 19.11 38 0.8235 20.79 39 0.3032 3.030 40 2.453 45.99 41 1.47136.96 42 0.4948 20.76 43 2.568 81.94 44 0.6136 13.77 45 2.502 17.58 4610.28 40.65 47 1.042 143.6 48 1.127 6.014 49 0.4841 4.322 Mean^(b) 2.50938.07 Median^(b) 1.406 18.07 Standard 2.970 56.92 Deviation^(b)Confidence 1.624-3.394 32.80-43.34 Interval^(b,c)

[0082] TABLE 2 Levels of β-alanine and β-aminoisobutyric acid in urinefrom a matched control group. β-Alanine^(a) β-Aminoisobutyric Acid^(a)Subject (μmol/mmol Creatinine) (μmol/mmol Creatinine)  1 0.2763 4.567  210.44 9.216  3 0.4764 3.560  4 4.736 11.44  5 0.7618 6.124  6 19.389.079  7 <0.1075 8.300  8 1.573 37.08  9 0.7881 9.435 10 6.375 8.064 110.4085 12.92 12 1.403 7.159 13 3.122 12.92 14 0.2249 8.300 15 1.66221.04 16 0.8533 3.811 17 0.7743 8.101 18 0.1502 7.979 19 1.806 60.68 200.6231 18.14 21 2.518 16.99 22 0.3880 36.44 23 2.921 46.43 24 <0.051044.627 25 0.4782 8.229 26 2.480 5.205 27 0.5029 21.74 28 1.248 22.37 2915.27 14.16 30 0.4514 3.354 31 43.41 25.95 32 0.9628 31.99 33 12.449.725 34 11.31 21.30 35 0.2259 3.219 36 0.2200 37.65 37 3.418 14.43 380.4532 26.93 39 0.6999 10.26 40 0.04266 18.26 41 0.1145 6.350 42 <0.11472.176 43 1.822 3.131 44 0.7089 4.340 45 0.1500 20.69 46 1.390 6.114 470.7200 38.42 48 2.612 15.40 49 8.350 15.50 50 <0.07333 9.588 51 8.90168.91 Mean^(b) 2.916 16.43 Median^(b) 8.533 10.26 Standard 4.390 14.63Deviation^(b) Confidence 1.626-4.206 12.29-20.57 Interval^(b,c)

[0083] Nonparametric statistical analysis was used to assess thestatistical significance of differences in urinary β-amino acidconcentrations observed between subjects with epilepsy and matchedcontrol subjects. Using a two-tailed Mann-Whitney test with a normalapproximation, a significant difference for β-aminoisobutyric acid(Zc=2.40, 0.01<P<0.02) was found between subjects with epilepsy andmatched control subjects.

[0084] The corresponding one-tailed tests showed that levels ofβ-aminoisobutyric acid were higher for those subjects with epilepsy(Zc=2.40, 0.005<P<0.01). Significant differences in β-alanine andβ-aminoisobutyric acid concentrations were not observed when comparingmale subjects with epilepsy to female subjects with epilepsy (β-alanineU=338; β-aminoisobutyric acid U=354) as well as when comparing malesubjects in the control population to female subjects in controlpopulation (β-alanine U=229; β-aminoisobutyric acid U=267). Theseresults show that gender does not influence the statistical significanceof the observed differences in urinary β-amino acid concentrationsbetween subjects with epilepsy and matched control subjects.

[0085] Using a Mann-Whitney test with a normal approximation,significant differences in urinary β-aminoisobutyric acid concentrations(Zc=2.37, 0.01<P<0.02) were determined upon comparing male subjects withepilepsy to male matched control subjects. Based on the correspondingone-tailed Mann-Whitney test, these levels were statistically higher forthose male subjects with epilepsy (Zc=2.37, 0.005<P<0.01).

[0086] Using a Krusal-Wallis test with a chi-square approximation,significant differences in the urinary levels of β-alanine andβ-aminoisobutyric acid were not detected between subgroups E+D+, E−D+and E−D− of the population with epilepsy (β-alanine Hc=0.585;β-aminoisobutyric acid H=0.266). Nor were differences determined betweensubgroups C-D+ and C−D− of the control population, using a two-tailedMann-Whitney test with a normal approximation (β-alanine Zc=0.387;β-aminoisobutyric acid Zc=0.670).

[0087] These results show that seizure frequency and anticonvulsantmedication do not affect the statistical significance of the observeddifferences in urinary β-amino acid concentrations. A comparison ofsubjects with epilepsy in groups E+D+ and E−D+, receiving anticonvulsantmedication, to matched control subjects in the group C−D−, not receivinganticonvulsant medication, demonstrated a significant difference forβ-aminoisobutyric acid (Zc=2.08, 0.02 <P<0.05), with higher levels ofthis amino acid occurring in the urine of these subjects with epilepsy(Zc=2.08, 0.01<P<0.025).

[0088] Similarly, a comparison of subjects with epilepsy in groups E+D+and E−D+, receiving anticonvulsant medication, to matched controlsubjects in the group C−D+, also receiving anticonvulsant medication,demonstrated significant differences in β-aminoisobutyric acid levels(Zc=2.06, 0.02<P<0.05). β-Aminoisobutyric acid concentrations were againfound to be statistically higher for these subjects with epilepsy, asdetermined by a one-tailed Mann-Whitney test with a normal approximation(Zc=2.06, 0.01 <P<0.025). These results show that significantdifferences in urinary β-aminoisobutyric acid concentrations are notinfluenced by anticonvulsant medication alone.

[0089] The decision to analyze urine rather than cerebrospinal fluid(CSF) or plasma was taken after careful consideration. It has long beenappreciated that the chemical milieu of the CSF provides informationregarding abnormal cerebral metabolism. CSF is not, however, a mereultrafiltrate formed by the choroids plexus, but arises from theinteractions between blood and the CNS (Perry, et al., Clin. Invest.1961, 40, 1363-1372). These interactions permit many substances,especially amino acids, to exhibit similar levels in plasma as well asin CSF.

[0090] Scriver et al. in their studies of people withhyper-β-alaninemia, clearly demonstrated that the increased levels ofβ-alanine in the CSF are directly reflected in plasma (op. cit.). Theyalso noted a direct relationship between plasma levels and urinaryexcretion, attributing this observation to the renal tubular transportof β-amino acids. Urinary levels of β-alanine therefore provide a windowof observation into the metabolism of β-alanine within the CNS.

[0091] From an analytical point of view, urine analysis is preferred asthe control range for β-aminoisobutyric acid in adult urine issubstantially higher when compared to plasma or CSF (β-aminoisobutyricacid=10-510 μmol/L for urine, 0 μmol/L for plasma, <10 nmol/L for CSF).Urine also represents an easily accessible biological fluid in which tocollect from the brain-injured individual.

[0092] To validate the notion of screening for seizure susceptibilitybased on urinary levels of β-alanine and β-aminoisobutyric acid, thesensitivity and specificity of this assay were calculated at definedβ-amino acid concentrations. Optimal results were achieved when thecut-offs for seizure susceptibility were set at concentrations of 0.8μmol/mmol creatinine for β-alanine and 10 μmol/mmol creatinine forβ-aminoisobutyric acid in urine samples, although useful clinical datamay be obtained at other cut-off values. The skilled artisan willappreciate that such cut-off levels may be different for other bodilyfluids. For β-aminoisobutyric, the sensitivity of this assay, defined asthe probability of testing positive for seizure susceptibility when asusceptibility is truly present, was determined to be 73%, while thespecificity, defined as the probability of testing negative for seizuresusceptibility when no susceptibility exists, was 47%

[0093] In summary, the results indicate that subjects with seizuredisorders excrete more β-aminoisobutyric acid in their urine than peoplewho do not have seizure disorders. Urinary levels of these amino acidswere statistically higher for the 49 subjects with epilepsy relative tothe 51 matched control subjects. Statistical differences are notsignificantly influenced by gender, administration of anticonvulsantmedication, or seizure frequency. Accordingly urinary concentrations ofβ-alanine and β-aminoisobutyric acid may be used as biological markersfor seizure presence and susceptibility and epileptogenesis.

[0094] The potential clinical applications of measurements of urinaryβ-alanine and β-aminoisobutyric acid levels are multiple. First, such anassay may assist in verifying the presence of epilepsy. Differentiatingseizures from nonepileptic seizures (psuedoseizures) is a commonclinical problem. Urinary β-aminoisobutyric acid levels augment clinicalobservation, EEG studies and serum prolactin measurements as usefulclinical tools in the differentiation between epileptic and nonepilepticseizures. Secondly, a urinary assay for β-aminoisobutyric acid hasutility in predicting seizure susceptibility. Seizures may arise from adiversity of CNS insults, including trauma, infection, ischaemia, andneoplasia. Identifying which subset of patients will ultimately developrecurrent seizures after such an insult is currently an unattainableclinical goal. The predictive test described herein to identify thosewith a predisposition to epilepsy therefore has significant clinicalvalue.

Equivalents

[0095] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims. The contents of all references, issued patents, and publishedpatent applications cited throughout this application are herebyincorporated by reference.

What is claimed is:
 1. A method of diagnosing a convulsive condition orsusceptibility thereto in a subject comprising the steps of a. analyzinga bodily fluid from a subject for the presence or amount of aneuro-active molecule, or the relative amounts of neuro-activemolecules, associated with a convulsive condition; and b. diagnosing thesubject as at risk of a convulsive condition or susceptibility theretowhen the amount of said compound indicates a likelihood of same in saidsubject.
 2. A method of modulating the onset of a convulsive conditionin a subject comprising the steps of analyzing a bodily fluid from asubject at risk of a convulsive condition for the presence or amount ofa neuro-active molecule, or the relative amounts of neuro-activemolecules, associated with a convulsive condition; determining from theamount of said compound in said bodily fluid whether said subject is atrisk of a convulsive condition; and treating said subject, if at risk ofa convulsive condition, to modulate the onset of said convulsivecondition in said subject.
 3. A method of diagnosing a convulsivecondition or susceptibility thereto in a subject comprising the steps ofanalyzing a bodily fluid from a subject for the presence or amount of aβ-amino acid, or the relative amount of β-amino acid; and diagnosing thesubject as at risk of a convulsive condition when the amount of saidβ-amino acid indicates a likelihood of same in said subject.
 4. A methodof modulating the onset of a convulsive condition in a subjectcomprising the steps of analyzing a bodily fluid from a subject at riskof a convulsive condition for the presence or amount of a β-amino acid,or the relative amount of β-amino acid; determining from the amount ofsaid β-amino acid in said bodily fluid whether said subject is at riskof a convulsive condition, and treating said subject, if at risk of aconvulsive condition, so as to modulate the onset of said convulsivecondition in said subject.
 5. The method of any claim herein, whereinsaid neuro-active molecule is a metabolite of uracil or thymine, or aderivative thereof.
 6. The method of any claim herein, wherein saidcompound is an amino acid or a derivative thereof.
 7. The method ofclaim 5, wherein said metabolite is a β-amino carboxylic acid.
 8. Themethod of any one of claims 5, 6, or 7, wherein said β-amino acid isselected from β-aminoisobutyric acid and derivatives thereof.
 9. Themethod of any claim herein, wherein said convulsive condition isselected from the group consisting of epileptogenic associateddisorders, epileptogenesis, and non-epileptic convulsions.
 10. Themethod of any claim herein, wherein said convulsive condition is anepileptogenic-associated disorder selected from epilepsy, head trauma,stroke, multiple sclerosis, amyotrophic lateral sclerosis, psychoses,cerebral ischemia, motor neuron disease, Alzheimer's disease,chicken-pox, measles, encephalitis, pertussis encephalitis, infectionsof the CNS, meningitis, encephalitis, subdural haematoma, brain tumour,birth defects, anoxic brain injury dementia, or other disorders in whichaltered activity of neuro-active molecules is a cause, at least in part,of the disorder.
 11. The method of any claim herein, wherein saidconvulsive condition is selected from the group consisting of epilepsyand non-epileptic convulsions.
 12. The method of any claim herein,wherein said subject has not yet developed seizures.
 13. The method ofany claim herein, wherein said treatment step comprises administering aneffective amount of an anti-convulsive pharmaceutical composition. 14.The method of any claim herein, wherein said treatment step comprisesadministering an effective amount of an anti-epileptogenicpharmaceutical composition.
 15. The method of any claim herein, furthercomprising the steps of deproteinizing said bodily fluid.
 16. The methodof claim 15, wherein said deproteinizing step is ultrafiltration,ultracentrifugation, or chemical precipitation.
 17. The method of claim16, wherein said chemical precipitation step employs sulfosalicylicacid, perchloric acid, trichloroacetic acid, picric acid, acetonitrile,ethanol, acetone, or methanol.
 18. The method of any claim herein,further comprising derivatizing said amino acid prior to analyzing it.19. The method of claim 18, wherein said derivatizing step covalentlyattaches a chromophore to said amino acid.
 20. The method of claim 18,wherein said derivatizing step employs o-phthalaldehyde,9-fluorenylmethylchloroformate, phenyl isothiocyanate, or1-dimethylaminonaphthalene-5-sulphonyl chloride.
 21. The method of anyclaim herein, wherein said bodily fluid is urine, blood, plasma, bloodserum, cerebrospinal fluid, sweat, lymph, amniotic fluid, synovialfluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminalfluid, bile, tears, or mixtures thereof.
 22. The method of claim 20,wherein said bodily fluid is urine.
 23. The method of claim 13, whereinsaid anti-seizure pharmaceutical composition is carbamazepine, clobazam,diazepam, lamotrigine, lorazepam, oxazepam, phenobarbital, phenytoin,primidone, valproate, ethosuximide, topirimate, felbamate, clonazepam,clobazam, nitrazepam, vigabatrin, gabapentin, levetiracetam, zonasimide,or tiagabine, or combinations thereof.
 24. The method of any claimherein, wherein said subject is an animal or human.
 25. The method ofclaim 24, wherein said subject has a family history of convulsiveconditions, a history of cerebral hypoxia or ischemia, intracranialhemorrhage, central nervous system infection or disease, drug or alcoholwithdrawal, fever, trauma, brain tumor, cerebrovascular disease,metabolic disorder, degenerative central nervous system disease, drug oralcohol addiction or use, uremia, hepatic dysfunction, hypoglycemia,epilepsy, or seizure; or said patient has recently been administered anantibiotic, anesthetic, analgesic, immunomodulatory, psychotropic,sedative, antihistamine, radiographic contrast-enhancing, stimulant orhallucinogenic drug.
 26. The method of any claim herein, wherein saidsubject has suffered a head trauma.
 27. The method of claim 26, whereinsaid convulsive condition is post-traumatic epilepsy (PTE) or asusceptibility thereto.
 28. The method of any claim herein, wherein saidseizure is selected from the group consisting of complex partial, simplepartial, absence, secondary generalized tonic clonic, primarygeneralized tonic clonic, myoclonic, and atonic.
 29. The method of anyclaim herein, wherein the analyzing step comprises chromatography,spectroscopy, spectrometry, or colorimetry.
 30. The method of claim 29,wherein said chromatography is high-performance liquid chromatography,thin layer chromatography, or gas chromatography.
 31. The method of anyclaim herein, wherein said treating is carried out when the amount ofsaid amino acid is substantially different from the average amountpresent in a matched control population.
 32. The method of claim 28,wherein the amount of amino acid in the urine is statisticallysignificantly higher than in a matched control population.
 33. Themethod of claim 32, wherein said amino acid is β-aminoisobutyric acid.34. A method of quantifying β-alanine or β-aminoisobutyric acidcomprising the steps of collecting a urine sample; deproteinizing saidurine sample; derivatizing the amino acids present in said deproteinizedurine sample; and analyzing said derivatized amino acids by highperformance liquid chromatography, said chromatography system comprisinga reversed phase column, acetate buffer and methanol mobile phases, aninternal standard, and a separation program which produces a resolutionfor each of β-alanine and β-aminoisobutyric acid with all other aminoacids and molecules present in said urine of equal to or greater thanone.
 35. The method of claim 34, wherein said internal standard isD,L-ethionine.
 36. The method of claim 35, wherein said reversed phasecolumn is an C₈ or C₁₈ column.
 37. A kit for use according to theinvention comprising internal or external standards or derivatizingreagents, and instructions for use in the method of any claim herein.38. The kit according to claim 37, further comprising a chromatographysystem.
 39. The kit according to claim 38, wherein said chromatographysystem is a HPLC system.
 40. The method of any claim herein, whereinsaid step of determining or diagnosing is positive when the urinaryconcentration of β-alanine is greater than 0.8 μmol/mmol creatinine orβ-aminoisobutyric acid is greater than 10 μmol/mmol creatinine.